CIC-DUX4 Oncoprotein Drives Sarcoma Metastasis and Tumorigenesis Via Distinct Regulatory Programs
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CIC-DUX4 oncoprotein drives sarcoma metastasis and tumorigenesis via distinct regulatory programs Ross A. Okimoto, … , Tadashi Kondo, Trever G. Bivona J Clin Invest. 2019;129(8):3401-3406. https://doi.org/10.1172/JCI126366. Concise Communication Oncology Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua–double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4– expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4– expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications. Find the latest version: https://jci.me/126366/pdf The Journal of Clinical Investigation CONCISE COMMUNICATION CIC-DUX4 oncoprotein drives sarcoma metastasis and tumorigenesis via distinct regulatory programs Ross A. Okimoto,1,2 Wei Wu,1 Shigeki Nanjo,1 Victor Olivas,1 Yone K. Lin,1 Rovingaile Kriska Ponce,1 Rieko Oyama,3 Tadashi Kondo,3 and Trever G. Bivona1,2,4 1Department of Medicine, 2Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA. 3Division of Rare Cancer Research, National Cancer Center Research Institute, Tokyo, Japan. 4Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA. Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua–double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4–expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4–expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications. Introduction functions as a transcriptional activator instead of a transcriptional The biological regulation of transcription factors and repressor pro- repressor (5, 6). This property suggests that the C-terminal DUX4 teins is an essential mechanism for maintaining cellular homeo- binding partner may confer neomorphic transcriptional regulatory stasis, and is often dysregulated in human cancer (1). Indeed, properties to CIC, while retaining WT CIC DNA binding specificity. chromosomal rearrangements involving transcriptional regula- ETV4 is one of the most well-characterized transcriptional targets tory genes that lead to transcriptional dysregulation are present of CIC-DUX4 (5, 7, 8). More than 90% of CIC-DUX4–expressing in many cancers, including approximately 30% of all soft tissue tumors show ETV4 upregulation by IHC, which distinguishes them sarcomas (2). The majority of these oncogenic fusions involve from other small round blue cell sarcomas (8). We and others have transcription factors or regulators that are not readily “drugga- demonstrated a prometastatic function for ETV4 overexpression in ble” in a direct pharmacologic manner and thus have proven dif- tumors with inactivation of WT CIC (9, 10). The functional role of ficult to therapeutically target in the clinic. A prime example is the ETV4 in CIC-DUX–positive tumors is unknown. CIC-DUX4 fusion oncoprotein, which fuses capicua (CIC) to the Beyond ETV4, the identity and function of other CIC target genes double homeobox 4 gene, DUX4. CIC-DUX4–positive soft tissue are less well defined. Intriguingly, recent studies showed increased tumors are an aggressive subset of undifferentiated round cell sar- expression of cell-cycle-regulatory genes in CIC-rearranged sarco- comas that arise in children and young adults. Despite histological mas, although the functional relevance of this observation for onco- similarities to Ewing sarcoma, CIC-DUX4–positive sarcomas are genesis and cancer growth in this context is unclear (6, 7). clinically distinct and typically characterized by the rapid devel- Here, we developed a range of in vitro and in vivo cancer opment of lethal metastatic disease and chemoresistance (3). By model systems to define the mechanism by which CIC-DUX4 revealing downstream molecular targets that relay the functional controls capicua-regulated transcriptional pathways to promote output of CIC-DUX4, we can potentially identify and develop a hallmark features of malignancy, including tumor cell survival, more rational therapeutic strategy to improve patient outcomes. growth, and metastasis. CIC is a transcriptional repressor protein (4). The CIC-DUX4 fusion structurally retains greater than 90% of native CIC, yet it Results and Discussion We recently reported that inactivation of native CIC derepresses an ETV4-MMP24–mediated prometastatic circuit (9). Since ETV4 Conflict of interest: TGB is an advisor to Revolution Medicines, Novartis, AstraZen- is a direct transcriptional target of CIC-DUX4 (Supplemental Fig- eca, Takeda, SpringWorks, Jazz, and Array BioPharma and receives research funding ure 1A; supplemental material available online with this article; from Revolution Medicines and Novartis. https://doi.org/10.1172/JCI126366DS1) (6, 7), we hypothesized Copyright: © 2019, American Society for Clinical Investigation. that the high metastatic rate observed in patients harboring CIC- Submitted: November 21, 2018; Accepted: May 24, 2019; Published: July 22, 2019. Reference information: J Clin Invest. 2019;129(8):3401–3406. DUX4 fusions was dependent on ETV4 expression. To explore this https://doi.org/10.1172/JCI126366. hypothesis, we developed an orthotopic soft tissue metastasis mod- jci.org Volume 129 Number 8 August 2019 3401 CONCISE COMMUNICATION The Journal of Clinical Investigation Figure 1. ETV4 promotes metastasis in CIC-DUX4–expressing sarcoma. (A) Schematic of the orthotopic soft tissue metastasis model. (B) Representative bioluminescence (BLI) images from mice bearing CIC-DUX4–expressing NIH 3T3 cells with shCtrl (n = 9) or shETV4 (n = 7). (C) Immunoblot of ETV4 and MMP24 from NIH 3T3 cells expressing empty vector (EV), shCtrl, shETV4a, or shETV4b. (D) Number of lung metastases in mice bearing CIC-DUX4–express- ing NIH 3T3 cells with shCtrl (n = 9) or shETV4 (n = 7). P value, Student’s t test. (E) Transwell invasion assay comparing CIC-DUX4–expressing NIH 3T3 cells with EV, shCtrl, shETV4a, or shETV4b. P value, 1-way ANOVA. Error bars represent SEM. (F) Relative photon flux from mice orthotopically implanted with CIC-DUX4–expressing NIH 3T3 cells expressing either shCtrl or shETV4. Error bars reflect SEM. (G) Subcutaneously implanted NIH 3T3 cells with EV, CIC- DUX4, or CIC-DUX4 plus shETV4a or shETVb. P value, 1-way ANOVA. Error bars represent SEM. el utilizing luciferase-based imaging to track tumor dissemination ed that the CIC-DUX4 fusion can regulate cell cycle gene expres- in vivo (Figure 1A). This system produces rapid pulmonary metas- sion (6, 7). We established that ectopic expression of CIC-DUX4 tases, accurately recapitulating metastatic tumor dissemination increased NIH 3T3 cell growth in vitro and in vivo through enhanced in patients (3). Using this in vivo system, we engineered NIH 3T3 cell cycle progression (Supplemental Figure 2, A–E). The CIC-DUX4 mouse fibroblasts, which offer the advantage of a genetically con- fusion increased the number of cells progressing through S-phase, as trolled system (as with the study of other oncoproteins), to express reflected by an increased G2/M fraction compared with control (Sup- the CIC-DUX4 fusion oncoprotein (11–13). We observed rapid pri- plemental Figure 2F). In order to identify CIC-DUX4–regulated cell mary tumor formation at the site of implantation in 100% of the cycle genes, we leveraged a publicly available microarray-based data injected mice (Figure 1B). Mice with genetic silencing of ETV4 set (GSE60740) to perform a comparative transcriptional analysis showed decreased expression of its established target, MMP24 between CIC-DUX4–replete (IB120 EV cell line) and 2 independent (9) (Figure 1C, and Supplemental Figure 1, B and C), and impaired CIC-DUX4–knockdown (KD)